EP0698102B1 - Cholesterinoxidase aus brevibacterium sterolicum - Google Patents

Cholesterinoxidase aus brevibacterium sterolicum Download PDF

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Publication number: EP0698102B1
Authority: EP; European Patent Office
Prior art keywords: seq; cholesterol oxidase; dna; sequence; cholesterol
Prior art date: 1993-05-05
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Expired - Lifetime

Application number

EP94915569A

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German (de)

English (en)

French (fr)

Other versions

EP0698102A1 (de

Inventor

Michael Jarsch

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Roche Diagnostics GmbH

Original Assignee

Roche Diagnostics GmbH

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

1993-05-05

Filing date

1994-05-02

Publication date

2006-03-01

1993-12-09 Priority claimed from DE4342012A external-priority patent/DE4342012A1/de

1994-05-02 Application filed by Roche Diagnostics GmbH filed Critical Roche Diagnostics GmbH

1996-02-28 Publication of EP0698102A1 publication Critical patent/EP0698102A1/de

2006-03-01 Application granted granted Critical

2006-03-01 Publication of EP0698102B1 publication Critical patent/EP0698102B1/de

2014-05-02 Anticipated expiration legal-status Critical

Status Expired - Lifetime legal-status Critical Current

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Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide

Definitions

the invention relates to a cholesterol oxidase from Brevibacterium sterolicum, a method for producing a recombinant cholesterol oxidase from Brevibacterium sterolicum, a DNA sequence suitable for this method which causes cytoplasmic expression of the recombinant cholesterol oxidase in the host bacterium, and the recombinant cholesterol oxidase thus obtainable.
Cholesterol oxidase is of great importance for the enzymatic determination of cholesterol. It catalyzes the oxidation of cholesterol to cholesten-3-one and H 2 O 2 . Cholesterol oxidase from various organisms such as Pseudomonas, Mycobacterium, Nocardia, Arthrobacter and Brevibacterium have already been described (T. Uwajima et al., Agr. Biol. Chem. 37 (1973), 2345-2350). All of these known cholesterol oxidases are secreted proteins. The soil bacterium Brevibacterium sterolicum KY 3643 (ATCC 21387) shows a particularly high activity of cholesterol oxidase.
EP-A 0 452 112 describes the cloning and expression of further cholesterol oxidases from Brevibacterium sterolicum. However, the expression of these DNAs also does not result in a sufficient amount of active cholesterol oxidase.
the object of the invention was to provide a cholesterol oxidase with high affinity for cholesterol in large quantities and in active form.
This object is achieved by a cholesterol oxidase which has the amino acid sequence shown in SEQ ID NO 2.
This cholesterol oxidase is obtainable from Brevibacterium sterolicum or else produced recombinantly.
This cholesterol oxidase can be produced recombinantly in large quantity and in active form.
This cholesterol oxidase has a molecular weight of 60 kD, an isoelectric point of about 5.5 (each measured in the Phast system, Pharmacia-LKB) and a K M value for cholesterol of 1 x 10 -4 mol / l (in 0 , 5 mol / l potassium phosphate buffer pH 7.5 at 25 ° C) and is effective in a pH range of 5.5 to 8.0.
this cholesterol oxidase can be obtained in a large amount in an active form when a DNA coding for a peptide having cholesterol oxidase activity with the DNA sequence shown in SEQ ID NO 1 or the one shown in SEQ ID NO 1 is used for heterologous expression complementary DNA sequence.
a DNA having the sequence shown in SEQ ID NO 1 is used.
degenerate codons can be replaced by other codons which code for the same amino acid.
the DNA used should have one of the DNA sequences shown in SEQ ID NO 3, 4 and / or 5 and code for a peptide having cholesterol oxidase activity.
a peptide having cholesterol oxidase activity is meant such a peptide which catalyzes the oxidation of cholesterol (5-cholesten-3- ⁇ -ol) to 4-cholesten-3-one and H 2 O 2 .
Another object of the invention is therefore a DNA which codes for a peptide having cholesterol oxidase activity with the DNA sequence shown in SEQ ID NO 1 or the complementary DNA sequence.
Another object of the invention is a method for producing a recombinant cholesterol oxidase by transformation of a suitable host cell with a DNA of the invention, which is present in a suitable expression system, culturing the transformed host cells and isolation of the formed cholesterol oxidase from the cytoplasm of the transformed cells.
the DNA used at the 5 'end may contain an additional nucleotide sequence which has a translation start codon, but no stop codon, wherein this additional nucleotide sequence does not result in frameshifting and is not a functionally active signal sequence for secretion of the recombinant enzyme formed.
the length of this nucleotide sequence is about 3 to 90 base pairs.
the additional nucleotide sequence has one of the sequences shown in Sequence Listings 6, 8, 10, 12, 14 and 16 in place of the native signal sequence.
a preferred subject of the invention is therefore a process for the preparation of a recombinant cholesterol oxidase using a DNA according to the invention which has one of the sequences shown in SEQ ID NO 6, 8, 10, 12, 14 or 16 at the 5 'end.
the transformation of the host cells used for recombinant production is carried out by known methods (see, e.g., Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).
the transformed host cells are then cultured under conditions that allow expression of the cholesterol oxidase gene.
an inducer for example lactose or isopropyl- ⁇ -D-thiogalactopyranoside (IPTG)
IPTG isopropyl- ⁇ -D-thiogalactopyranoside
the isolation of the recombinant cholesterol oxidase from the cytoplasm of the transformed cells is then carried out by known methods.
the cholesterol oxidase of the present invention as a recombinant enzyme in a yield of 8-20 U / ml.
the expression of the complete the cholesterol oxidase gene containing the signal sequence gives only a yield of less than 0.1 U / ml.
a preferred subject matter of the invention is a DNA coding for a peptide having cholesterol oxidase activity according to the invention, which has one of the sequences shown in SEQ ID NO 6, 8, 10, 12, 14 and 16 at the 5 'end. Particularly preferred are the sequences shown in Sequence Listings 18, 20, 22, 24, 26 and 29.
these DNA sequences according to the invention are cloned in an expression vector.
the cholesterol oxidase according to the invention can be obtained in any amount in the bacteria commonly used for the recombinant production of proteins.
the expression takes place in E. coli.
Another object of the invention is therefore a recombinant cholesterol oxidase, which is encoded by a DNA according to the invention and having at the N-terminal end one of the amino acid sequences shown in SEQ ID NO 7, 9, 11, 13, 15 or 17.
This recombinant cholesterol oxidase is suitable for an enzymatic assay for the determination of cholesterol as well as the other known from the prior art cholesterol oxidases.
recognition sequences for specific proteases such as IgA protease, enterokinase or factor Xa can be integrated by in vitro mutagenesis between these N-terminal sequences and the amino acid sequence of the mature cholesterol oxidase, as known to those skilled in the art even after the cytoplasmic expression of extended to these N-terminal sequences cholesterol oxidase cleavage of such anfused N-terminal sequences is possible.
a preferred subject of the invention is a recombinant cholesterol oxidase having the amino acid sequence shown in SEQ ID NO 21, 23, 25, 27 or 29 and the use of such a recombinant cholesterol oxidase in an enzymatic assay for the detection of cholesterol.
the H 2 O 2 formed in the cholesterol oxidase reaction is preferably determined in a downstream indicator reaction as a measure of the cholesterol present in the sample.
the plasmids pUC-Chol-B2-BB (DSM 8274), pmgl-SphI (DSM 8272) and pfl-20AT1-SD (DSM 8273) mentioned in the examples were used on 05.05.1993 by the Deutsche Sammlung für Zellkulturen und Mikroorganismen GmbH, Mascheroder Weg 1b, D - 3300 Braunschweig deposited.
Brevibacterium sterolicum (BMTU 2407) is grown in 500 ml of nutrient broth (Difco) for 20 h at 30 ° C. The cells are harvested by centrifugation. The cell mass thus obtained is resuspended in 20 mmol / l Tris / HCl pH 8.0 to 0.4 g cell wet weight / ml. 2.5 ml of this suspension are mixed with 5 ml of 24% (w / v) polyethylene glycol 6000, 2.5 ml of 20 mmol / l Tris / HCl pH 8.0 and 10 mg of lysozyme and incubated at 4 ° C for 14 h.
the cells are lysed by adding 1 ml of 20% (w / v) SDS and 2 mg of protease K and incubating for 1 h at 37 ° C.
This solution comes with the same volume 20 mmol / l Tris / HCl pH 8.0 and then per ml 1 g CsCl and 0.8 mg ethidium bromide added.
This solution is separated by ultracentrifugation for 24 h at 40,000 rpm in a TV850 vertical rotor (DuPont).
the DNA band is then withdrawn with a hypodermic syringe.
the removal of the ethidium bromide and ethanol precipitation of the DNA is carried out as described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989).
the tall colonies are transferred to nitrocellulose filters (Schleicher and Schuell), lysed by treatment with toluene / chloroform vapor and the filters are transferred to the colony side on indicator plates (see below). On these indicator plates, the detection of cholesterol oxidase activity is carried out by incubation at room temperature for 15 to 30 minutes.
Clones showing a color reaction are selected and isolated.
these E. coli clones are spread on an agar plate with LB medium containing 100 .mu.g / ml ampicillin, incubated overnight at 37.degree. C., the grown colonies are transferred again to two different nitrocellulose filters for verification and as above described digested with toluene / chloroform vapor.
a filter is placed again on one of the indicator plates described above, the other moths on a indicator plate without cholesterol.
a positive color reaction only occurs on the complete indicator plates with the substrate cholesterol. This demonstrates that the color reaction caused by the corresponding E. coli clone is actually caused by active cholesterol oxidase.
the plasmid of a clone obtained according to Example 1 (pUC-Chol-B2) is isolated according to standard methods and subjected to restriction mapping with the restriction endonucleases BamHI, EcoRI, KpnI, XhoI, PstI. It turns out that a DNA fragment from the genome of Brevibacterium in the size of about 5.5 kb is inserted in the plasmid pUC-Chol-B2. By subcloning various partial fragments of this 5.5 kb piece and then determining the cholesterol oxidase activity of the E. coli clones obtained, the cholesterol oxidase gene can be concentrated to a BamHI fragment of 2.3 kb size.
the plasmid with this fragment is called pUC-Chol-B2-BB ( Figure 1).
the DNA sequence of this fragment is determined and assayed on a reading frame encoding cholesterol oxidase.
the sequence of this reading frame for the mature cholesterol oxidase is shown in SEQ ID NO 1.
This PCR fragment is cleaved with SphI and PstI and ligated together with a PstI EcoRI fragment from pUC-Chol-B2-BB, which contains the remaining portion of the cholesterol oxidase gene, into the SphI and EcoRI digested expression vector pmglSphI and so Vector pmgl-Chol-SB obtained.
the cholesterol oxidase gene contains an E. coli functional signal sequence from Salmonella typhimurium (described in WO 88/093773).
a DNA fragment of 432 bp size is generated from the plasmid plac_Chol_cyt using the oligonucleotides shown in SEQ ID NO 32 and 33, which contains a ClaI site in front of the ATG start codon.
This PCR fragment is cut with ClaI and PstI.
PstI and BamHI By treatment with the restriction endonucleases PstI and BamHI, a fragment with the remaining C-terminal portion of the cholesterol oxidase gene is further excised from the plasmid plac-Chol-cyt. Both fragments are simultaneously in the expression vector pfl 20AT1-SD digested with BamHI and ClaI.
the correct ligation product now contains the reading frame of the mature cholesterol oxidase, fused to the first ten codons of the lacZ 'gene from pUC19 under the control of the oxygen-regulated pfl promoter ( Figure 3). This plasmid is called ppfl-chol-cyt.
100 ng of the thus purified DNA fragment are spiked with 50 pmol of an adapter oligonucleotide having the sequence shown in SEQ ID NO 34 (where "N” is an equimolar mixture of all 4 bases) and incubated for 2 hours at 37 ° C. with T4 DNA. Treated ligase.
the mixture is then mixed with a mixture of 4 dNTP's (final concentration 0.125 mmol / l) and treated with Klenow DNA polymerase for 40 minutes at 37 ° C.
the resulting plasmid DNA is transformed into E. coli XL1-blue (Stratagene).
the plasmid pfl-MSN3H-Chol-cyt aterm is cut with ClaI and XhoI.
a DNA fragment of about 1.1 kb with the translation initiation region and the N-terminal portion of the cholesterol oxidase gene is isolated and ligated into the also claI and XhoI cut plasmid ppfl-Chol-cyt ( Figure 4).
the resulting plasmid is called ppfl-MSN3H-Chol-cyt.
the plasmids pUC-Chol-B2, pUC-Chol-B2-BB, pmgl-Chol-SB, plac-Chol-cyt, pfl-Chol-cyt, ppfl-MSN3H-Chol-cyt are each in E. coli K12 XL1- blue transformed.
the clones are each 15 hours at 30 ° C in LB medium containing 200 ug / ml ampicillin and the following other additives, attracted: Clones with the plasmids pUC-Chol-B2, pUC-Chol-B2-BB, plac-Chol-cyt, in which the cholesterol oxidase gene is under the control of the lacUV5 promoter additionally 1 mmol / l IPTG, the clone with the plasmid pmgl-Chol-SB with the glucose-repressed mgl promoter receives no further addition, clones with the plasmids ppfl-Chol-cyt, ppfl-MSN3H-Chol-cyt with the oxygen-regulated pfl promoter receive 0.4% glucose and are gassed in nitrogen gassed sealed serum bottles with the medium adjusted to pH 7.0 with KOH.
the cell density achieved is determined by photometric measurement of the turbidity at 420 nm.
the cells of 1 ml of culture broth are then sedimented by centrifugation in a microcentrifuge at 10,000 g and resuspended again in 0.5 ml of H 2 O bidistilled.
the cell disruption is carried out by 2 x 30 seconds sonication (Branson Sonfier, model 450, standard microtip, conical).
the cell extracts thus obtained are used after appropriate dilution in the following enzyme test: For this purpose are pipetted into quartz cuvettes: 3 ml of potassium phosphate buffer (0.5 mol / l, pH 7.5), the 0.4% Thesit® (Boehringer Mannheim GmbH , Catalog No.
0.1 ml cholesterol solution (0.4% cholesterol, 10% 1-propanol, 10% Thesit®), 0.02 ml H 2 O 2 (0.49 mol / l in redistilled water), it is mixed, after addition of 0.02 ml catalase (from bovine liver, 20 mg protein / ml, specific activity about 65,000 U / mg, Boehringer Mannheim GmbH, Catalog No. 0156744 immediately before measurement with ice-cold potassium phosphate buffer, the 0, 4% thesitol diluted to 0.075-0.15 U / ml), the solution was brought to a temperature of 25 ° C and then the reaction started by adding 0.05 ml of sample solution.
0.02 ml catalase from bovine liver, 20 mg protein / ml, specific activity about 65,000 U / mg, Boehringer Mannheim GmbH, Catalog No. 0156744 immediately before measurement with ice-cold potassium phosphate buffer, the 0, 4% thesitol diluted to 0.075-0.15 U /
Table 1 Clone / plasmid Cell density (E 420) Units per cell density Units per ml pUC-Chol-B2 7.0 0,007 0,049 pUC-Chol-B2-BB 8.4 0,068 0.571 PMGL-Chol-SB 1.3 0,014 0,018 plac-Chol cyt 8.6 0.725 6,235 ppfl-Chol cyt 1.25 1.675 2,094 ppfl-MSN3H-Chol cyt 3.7 1,463 5,413

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EP94915569A 1993-05-05 1994-05-02 Cholesterinoxidase aus brevibacterium sterolicum Expired - Lifetime EP0698102B1 (de)

Applications Claiming Priority (5)

Application Number	Priority Date	Filing Date	Title
DE4314793		1993-05-05
DE4314793		1993-05-05
DE4342012A DE4342012A1 (de)	1993-05-05	1993-12-09	Cholesterinoxidase aus Brevibacterium sterolicum
DE4342012		1993-12-09
PCT/EP1994/001394 WO1994025603A1 (de)	1993-05-05	1994-05-02	Cholesterinoxidase aus brevibacterium sterolicum

Publications (2)

Publication Number	Publication Date
EP0698102A1 EP0698102A1 (de)	1996-02-28
EP0698102B1 true EP0698102B1 (de)	2006-03-01

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ID=25925592

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
EP94915569A Expired - Lifetime EP0698102B1 (de)	1993-05-05	1994-05-02	Cholesterinoxidase aus brevibacterium sterolicum

Country Status (8)

Country	Link
US (1)	US5916759A (fi)
EP (1)	EP0698102B1 (fi)
JP (1)	JP3506434B2 (fi)
AT (1)	ATE318910T1 (fi)
DE (1)	DE59410428D1 (fi)
DK (1)	DK0698102T3 (fi)
FI (1)	FI955286L (fi)
WO (1)	WO1994025603A1 (fi)

Cited By (1)

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US8729099B2 (en)	2001-01-12	2014-05-20	Actelion Pharmaceuticals Ltd.	Pharmaceutically active piperidine derivatives

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* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US5602017A (en) *	1990-04-10	1997-02-11	Kyowa Hakko Kogyo Co., Ltd.	Cholesterol oxidase
ATE364718T1 (de) *	1997-04-01	2007-07-15	Solexa Ltd	Verfahren zur vervielfältigung von nukleinsäure
AU2003246942B2 (en)	2002-07-17	2009-06-25	Idorsia Pharmaceuticals Ltd	Piperidinetriol derivatives as inhibitors of glycosylceramide synthase
GB0313677D0 (en)	2003-06-13	2003-07-16	Oxford Glycosciences Uk Ltd	Novel compound
GB0313678D0 (en)	2003-06-13	2003-07-16	Oxford Glycosciences Uk Ltd	Novel compounds
CN102380347B (zh) *	2010-09-06	2014-01-29	江南大学	一种胆固醇氧化酶亲和介质及其合成和大规模纯化胆固醇氧化酶方法
CN114606220B (zh) *	2020-11-25	2023-09-05	湖南引航生物科技有限公司	固定化经修饰的7α-羟基甾体脱氢酶及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US4374930A (en) *	1981-10-19	1983-02-22	Eastman Kodak Company	Method for the purification of cholesterol oxidase
JPH04218367A (ja) *	1990-04-10	1992-08-07	Kyowa Hakko Kogyo Co Ltd	新規コレステロール・オキシダーゼ
US5602017A (en) *	1990-04-10	1997-02-11	Kyowa Hakko Kogyo Co., Ltd.	Cholesterol oxidase

1994
- 1994-05-02 DE DE59410428T patent/DE59410428D1/de not_active Expired - Fee Related
- 1994-05-02 EP EP94915569A patent/EP0698102B1/de not_active Expired - Lifetime
- 1994-05-02 AT AT94915569T patent/ATE318910T1/de not_active IP Right Cessation
- 1994-05-02 US US08/532,795 patent/US5916759A/en not_active Expired - Fee Related
- 1994-05-02 WO PCT/EP1994/001394 patent/WO1994025603A1/de active IP Right Grant
- 1994-05-02 JP JP52390294A patent/JP3506434B2/ja not_active Expired - Fee Related
- 1994-05-02 DK DK94915569T patent/DK0698102T3/da active
1995
- 1995-11-03 FI FI955286A patent/FI955286L/fi unknown

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US8729099B2 (en)	2001-01-12	2014-05-20	Actelion Pharmaceuticals Ltd.	Pharmaceutically active piperidine derivatives
US9199935B2 (en)	2001-01-12	2015-12-01	Acetelion Pharmaceuticals Ltd.	Pharmaceutically active piperidine derivatives

Also Published As

Publication number	Publication date
JPH08510905A (ja)	1996-11-19
FI955286L (fi)	1996-01-03
DK0698102T3 (da)	2006-07-03
US5916759A (en)	1999-06-29
EP0698102A1 (de)	1996-02-28
DE59410428D1 (de)	2006-04-27
WO1994025603A1 (de)	1994-11-10
ATE318910T1 (de)	2006-03-15
FI955286A0 (fi)	1995-11-03
JP3506434B2 (ja)	2004-03-15

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